Prostate cancer detection methods

Abstract

The present invention provides methods of detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, and/or prognostication of prostate cancer comprising determining the average methylation ratio at 10 or more genomic regions as set out in the application, and associated methods of selecting a treatment or ascertaining whether a treatment is effective. The present invention also provides a method for determining a solid cancer circulating free DNA (cfDNA) methylome signature for use in the detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, and/or prognostication of the solid cancer in a sample obtained from a subject comprising determining the average methylation ratio at 10 or more genomic regions as set out in the application.

1 . A method for detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, and/or prognostication of prostate cancer in a sample obtained from a subject, wherein the sample comprises circulating free DNA (cfDNA), the method comprising:
 characterizing the methylome sequence of a plurality of cfDNA molecules in the sample, wherein the methylome sequence of a cfDNA molecule is the DNA sequence and the methylation profile of the molecule;   determining the average methylation ratio at 10 or more genomic regions, each genomic region being selected from the group consisting of:   a 100 to 200 bp region comprising or having a genomic location defined in Tables 1 to 4, and   a 2 to 99 bp region within a genomic location defined in Tables 1 to 4 and comprising at least one CpG locus,   and wherein each of the genomic regions is covered by at least one sequence read of at least one characterized methylome sequence;   calculating a methylation score using the average methylation ratio for each of the genomic regions;   analyzing the methylation score to determine the level of prostate cancer fraction in the cfDNA sample.   
     
     
         2 . The method of  claim 1 , wherein each of the genomic regions is covered by at least one sequence read of at least two characterized methylome sequences, for example at least one sequence read of at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 50, 100, 200, 300, 400, 500, or 1000 characterized methylome sequences. 
     
     
         3 . The method of  claim 1  or  2 , wherein each of the genomic regions is covered by at least 10 sequence reads, for example at least 10, 12, 15, 20, 25, 50, 100, 200, 300, 400, 500, or 1000 sequence reads, and preferably wherein each sequence read or the majority of the sequence reads (for example at least 50%, 60%, 70%, 80% or 90% of the sequence reads) are from different characterized methylome sequences. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein calculating a methylation score using the average methylation ratio for each genomic region comprises:
 determining the median (or the mean) of the average methylation ratios for all genomic regions for which the average methylation ratio has been determined; or 
 determining the median (or the mean) of the average methylation ratios for a first group of genomic regions to obtain a first methylation score and/or determining the median (or the mean) of the average methylation ratios for second group of genomic regions to obtain a second methylation score; or 
 comparing the average methylation ratio at each genomic region to a reference methylation ratio for each genomic region to determine a methylation ratio score for each genomic region. 
 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein analyzing the methylation score to determine the level of prostate cancer fraction in the cfDNA sample comprises comparing the methylation score to one or more reference methylation scores, wherein a reference methylation score is a methylation score calculated for the same genomic regions (for example, calculated using the average methylation ratio for the same genomic regions) in one or more of the following
 a cfDNA sample from a healthy subject, for example a healthy age-matched subject; 
 a tissue sample from a healthy subject, for example a prostate tissue sample from a healthy subject; 
 a cancer biopsy sample from a cancer patient, for example a prostate cancer biopsy sample from a prostate cancer patient; 
 a cancer cell line sample, for example a prostate cancer cell line sample from a prostate cancer cell line; 
 a sample of white blood cells from a subject, for example the subject or a healthy subject; 
 a cfDNA sample from a different subject having prostate cancer, preferably wherein the level of prostate cancer fraction in the cfDNA sample from the different subject is known (more preferably multiple cfDNA samples (for example at least 2, 3, 4, 5, 10, 20, 40, 50, 100, 200, 300 or 500 samples) each from a different subject having prostate cancer, wherein preferably the level of prostate cancer fraction in each cfDNA sample from the different subjects is known, and more preferably wherein each cfDNA sample has a different level of prostate cancer fraction); 
 a characterized methylome sequence of a white blood cell; 
 a characterized methylome sequence of a prostate cancer cell line; 
 a characterized methylome sequence of a cancerous prostate cell; and/or 
 a characterized methylome sequence of a non-cancerous prostate cell. 
 
     
     
         6 . The method of any one of  claims 1  to  5 , comprising determining the average methylation ratio at 25 or more, 50 or more, 100 or more, 150 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, or 900 or more genomic regions (for example comprising determining the average methylation ratio at 25, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 genomic regions). 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein the genomic regions have a 100 bp genomic location defined in any one of Tables 1 to 4, Table 5, Table 6 or Table 7. 
     
     
         8 . The method of any one of  claims 1  to  7 , wherein at least 25% of the genomic regions are prostate tissue specific genomic regions. 
     
     
         9 . The method of any one of  claims 1  to  8 , wherein the prostate cancer is acinar adenocarcinoma prostate cancer, ductal adenocarcinoma prostate cancer, transitional cell cancer of the prostate, squamous cell cancer of the prostate, or small cell prostate cancer (for example wherein the prostate cancer is acinar adenocarcinoma prostate cancer or ductal adenocarcinoma prostate cancer). 
     
     
         10 . The method of any one of  claims 1  to  9  wherein the prostate cancer is castration resistant prostate cancer and/or is metastatic prostate cancer. 
     
     
         11 . The method of any one of  claims 1  to  10 , wherein the sample comprising cfDNA is a blood or plasma sample. 
     
     
         12 . The method of any one of  claims 1  to  11 , further comprising repeating the method on a second sample obtained from the subject after the subject has undergone a treatment for prostate cancer, wherein the second sample comprises cfDNA, and comparing the level of prostate cancer fraction in the two samples. 
     
     
         13 . The method of any one of  claims 1  to  12 , further comprising treating the subject for prostate cancer using a therapeutic agent for the treatment of prostate cancer;
 or ceasing or altering treatment with a therapeutic agent for the treatment of prostate cancer; or 
 initiating a non-therapeutic agent treatment for prostate cancer (for example initiation of treatment by surgery or radiation). 
 
     
     
         14 . An in-vitro diagnostic kit for use in the detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, and/or prognostication of prostate cancer, comprising one or more reagents for detecting the presence or absence of at least 10 DNA molecules having a DNA sequence corresponding to all or part of a genomic location comprising at least one CpG locus defined in Tables 1 to 4, or comprising at least one CpG locus defined in Table 5, or comprising at least one CpG locus defined in Table 6, or comprising at least one CpG locus defined in Table 7. 
     
     
         15 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to perform the method of any one of  claims 1  to  12 ; or a computer-executable software for performing the method of any one of  claims 1  to  12  or a computer-implemented method for detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, and/or prognostication of prostate cancer in a sample obtained from a subject, wherein the sample comprises circulating free DNA (cfDNA), the method comprising:
 receiving a data set in a computer comprising a processor and a computer readable medium, 
 wherein the data set comprises the methylome sequence of a plurality of cfDNA molecules in the sample; 
 and wherein the computer readable medium comprises instructions that, when executed by the processor, causes the computer to perform a method of any one of  claims 1  to  12   
 
     
     
         16 . A therapeutic agent for the treatment of prostate cancer for use in the treatment of prostate cancer, whereby
 i) the method of any one of  claims 1  to  12  is performed to determine the level of prostate cancer prostate cancer DNA in a subject;   ii) the therapeutic agent is administered if the subject has a level of prostate cancer.   
     
     
         17 . A method of determining one or more suitable therapeutic agents for the treatment of prostate cancer for a subject having prostate cancer comprising
 performing the method of any one of  claims 1  to  12 ;   determining the one or more suitable therapeutic agents for the treatment of prostate cancer by reference to the level of prostate cancer, whereby one therapeutic agent is suitable for a subject with no level of prostate cancer fraction (for example an undetectable level of prostate cancer fraction) or a level of prostate cancer fraction of less than 0.01%, and two or more therapeutic agents are suitable for a subject with a level of prostate cancer DNA (for example a percentage level of prostate cancer fraction of at least 0.01%);   or whereby a therapeutic agent selected from a first list of therapeutic agents is suitable for a subject with no level of prostate cancer DNA (for example an undetectable level of prostate cancer DNA) or a level of prostate cancer DNA of less than 0.01%, and a therapeutic agent from a second list of therapeutic agents, or two or more therapeutic agents from the first list, is suitable for a subject with a level of prostate cancer DNA (for example a percentage level of prostate cancer fraction of at least 0.01%).   
     
     
         18 . A method or therapeutic agent as claimed in any one of  claim 16  or  17 , wherein the therapeutic agent for the treatment of prostate cancer is selected from the group consisting of a hormonal agent, a targeted agent, a biologic agent, an immunotherapy agent, a chemotherapy agent. 
     
     
         19 . A method for determining a solid cancer circulating free DNA (cfDNA) methylome signature for use in detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, prognostication and/or treatment of the solid cancer, the method comprising:
 (i) characterizing the methylome sequence of a plurality of cfDNA molecules in a first sample comprising cfDNA from a subject known to have the solid cancer, wherein the methylome sequence of a cfDNA molecule is the DNA sequence and the methylation profile of the molecule;   (ii) determining the respective number of characterised cfDNA molecules corresponding to a CpG locus or a genomic region of 2 to 10,000 bp (preferably 2 to 200 bp) in the first sample by aligning the methylome sequences;   (iii) determining the methylation ratio of each CpG locus and/or average methylation ratio of each genomic region of 2 to 10,000 bp (preferably 2 to 200 bp) in the first sample;   repeating steps (i) to (iii) for one or more further samples comprising cfDNA each from subjects known to have the solid cancer;   performing a variance analysis of all or a selection of the methylation ratios of the CpG loci and/or all or a selection of average methylation ratios of the genomic regions of the samples;   selecting a group of CpG loci and/or genomic regions associated with a feature of the samples;   selecting CpG loci and/or genomic regions in the group to provide the cfDNA methylome signature.   
     
     
         20 . The method of  claim 19 , wherein the solid cancer is prostate cancer. 
     
     
         21 . The method of  claim 19  or  20 , wherein the variance analysis performed is a dimensionality reduction. 
     
     
         22 . The method as claimed in  claim 21  wherein the variance analysis performed is a principal component analysis. 
     
     
         23 . The method as claimed in  claim 22 , wherein selecting a group of CpG loci and/or genomic regions associated with a feature of the samples comprises selecting one of principal component 1, principal component 2, principal component 3, principal component 4, principal component 5, principal component 6, principal component 7, principal component 8 or a higher principal component. 
     
     
         24 . The method of any one of  claims 18  to  23 , wherein selecting the CpG loci and/or genomic regions in the group to provide the cfDNA methylome signature comprises selecting the CpG loci and/or genomic regions in the group that have strong association with the feature, for example selecting CpG loci and/or genomic regions that are within the top 10,000 CpG loci and/or genomic regions most correlated with the feature in the group (for example selecting CpG loci and/or genomic regions that are within the top 8000, 5000, 3000, 2000, 1000, 800, 500, 400, 300, 250, 200, 150, 100, 50 or 10 CpG loci and/or genomic regions most correlated with the feature in the group). 
     
     
         25 . The method of any one of  claims 18  to  24 , wherein selecting CpG loci and/or genomic regions in the group to provide the cfDNA methylome signature comprises selecting at least 5 CpG loci (for example at least 8, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 or at least 10,000) and/or at least 5 genomic regions (for example at least 8, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 or at least 10,000) in the group to provide a cfDNA methylome signature. 
     
     
         26 . The method of  claim 22  or  23 , or  claim 24  or  25  when dependent on  claim 22  or  23 , wherein selecting CpG loci and/or genomic regions in the group to provide the cfDNA methylome signature comprises selecting a plurality of CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8, for example selecting CpG loci and/or genomic regions that are within the top 10,000 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8; or selecting CpG loci and/or genomic regions that are within the top 5000 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8;
 selecting CpG loci and/or genomic regions that are within the top 4000 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8; selecting CpG loci and/or genomic regions that are within the top 3000 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8; selecting CpG loci and/or genomic regions that are within the top 2000 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8; 
 selecting CpG loci and/or genomic regions that are within the top 1000 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8; or selecting CpG loci and/or genomic regions that are within the top 500, 400, 300, 250, 200, 150, 100, 50 or 10 CpG loci and/or genomic regions of principal component 1, 2, 3, 4, 5, 6, 7 or 8 most correlated with the feature of principal component 1, 2, 3, 4, 5, 6, 7 or 8. 
 
     
     
         27 . A method for detecting, screening, monitoring, staging, classification, selecting treatment for, ascertaining whether treatment is working in, and/or prognostication of prostate cancer in a sample obtained from a subject, wherein the sample comprises circulating free DNA (cfDNA), the method comprising:
 characterizing the methylome sequence of a plurality of cfDNA molecules in the sample, wherein the methylome sequence of a cfDNA molecule is the DNA sequence and the methylation profile of the molecule;   determining the average methylation ratio at 10 or more genomic regions, each genomic region being selected from the group consisting of:   a 100 to 200 bp region comprising or having a genomic location defined in Table 8, and   a 2 to 99 bp region within a genomic location defined in Table 8 and comprising at least one CpG locus,   and wherein each of the genomic regions is covered by at least one sequence read of at least one characterized methylome sequence;   calculating a methylation score using the average methylation ratio for each of the genomic regions;   analyzing the methylation score to determine whether the sample comprises cfDNA derived from a prostate cancer subtype.   
     
     
         28 . The method of  claim 27 , wherein the prostate cancer is an androgen-insensitive subtype of the prostate cancer.